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Systematic Evaluation of crRNA Design Parameters for Optimized Cas13d-Mediated RNA Targeting in Chicken Cells

ABSTRACT: CRISPR/Cas13 systems are increasingly becoming popular tools for programmable RNA targeting owing to its high-precision and ease at which crRNAs can be designed against mRNA targets of interest, often surpassing the usability of traditional RNA-targeting tools such as RNAi. The RNA-targeting capabilities of CRISPR/Cas13 have been harnessed to correct pathogenic mutations and combat infectious diseases. However, the wider adoption of Cas13 systems relies on the ability to design effective crRNAs that are broadly targetable and resistant to emerging mutants. In this study, we delineated the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for crRNAs, we designed multiple versions of crRNAs to investigate the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data revealed variable knockdown levels between crRNAs, in which a few crRNAs achieved over 95% target knockdown. We showed that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of the position at which the single-nt mismatch was introduced. However, 4-nt mismatches within crRNAs significantly reduced targeting efficacy, and eight nucleotide mismatches completely diminished targeting efficacy. Finally, we compared targeting efficiency of two widely used Cas13d variants (RfxCas13d and HfCas13d) and associated collateral degradation of bystander RNA. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.

ORGANISM(S): Gallus gallus

PROVIDER: GSE305666 | GEO | 2025/08/26

REPOSITORIES: GEO

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Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs

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Systematic Evaluation of crRNA Design Parameters for Optimized Cas13d-Mediated RNA Targeting in Chicken Cells

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